Transcription repressor

ABSTRACT

The present invention provides a human transcription repressor (TRAREP) and polynucleotides which identify and encode TRAREP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. In addition, the invention provides methods for producing TRAREP and for treating or preventing disorders associated with expression of TRAREP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of a newtranscription repressor and to the use of these sequences in thediagnosis, prevention, and treatment of inflammation and disordersassociated with cell proliferation and apoptosis.

BACKGROUND OF THE INVENTION

Transcription is a process by which genetic information encoded in DNAis transcribed into RNA. RNA polymerases I, II, and III are involved inthe process of transcription. RNA polymerase I is localized innucleosomes and is responsible for synthesis of the precursors of 28S,5.8S, and 18S rRNAs. RNA polymerase II transcribes genes encodingproteins and produces small RNAs responsible for RNA splicing. RNApolymerase III functions in conjunction with the nucleolus andtranscribes genes coding for tRNAs, 5S rRNA, and a series of small,stable RNAs.

Transcription of genes encoding proteins by RNA polymerase II isinitiated on DNA sequences corresponding to the 5' cap of mRNAs. Ahighly conserved, 25-35 base pair DNA sequence, TATA box, is positionedupstream of the transcription start site in most eukaryotic genes. Thefunction of the TATA box is to act as a promoter for transcription. RNApolymerase II and a series of transcription factors (TFIIA throughTFIIH) form a transcription initiation complex which binds at the TATAbox promoter region. The formation of this complex positions RNApolymerase II for the initiation of transcription.

The assembly of the transcription initiation complex begins with thebinding of TFIID to the TATA box. The process continues by theassociation of the TFIIA-TFIID-TATA complex with the remainingtranscription factors in the sequence of TFIIB, TFIIF-RNA polymerase II,TFIIE, TFIIH, and TFIIJ. Inhibitors or repressors that bind to theTFIID-promoter complex can potentially block further assembly of thetranscription initiation complex. Binding of TFIIA to the TFIID-TATAcomplex prevents the binding of the inhibitors and allows the assemblyto proceed.

Inhibition of transcription by repressor molecules is an important meansfor regulating transcription. Negative cofactor 2 (NC2) andDr1-associated polypeptides (DRAP1 and Dr1) are two such repressorswhich regulate transcription in cells. NC2 consists of two subunits,termed NC2α and NC2β, and the latter is identical to Dr1 (Goppelt, A. etal. (1996) EMBO J. 15: 3105-3116) in primary sequence. NC2α and NC2βpossess a histone fold domain of the H2A-H2B type at the amino terminusand an acidic domain at the carboxy terminus. NC2α has a proline-richdomain and a second, internal acidic domain, and NC2β has a basic domainand a Gln/Ala-rich domain. NC2 binds the TFIID-TATA promoter complex viathe histone fold domains and directly inhibits the binding of TFIIB withthe initiation complex. Repression of basal transcription requires thehistone fold domains and both of the acidic carboxy-terminal domains ofNC2 subunits. In nucleosomes, the histone fold effects dimerization andDNA-binding.

Similarly, DRAP1 and Dr1 form a heterodimer through the histone folddomains at the amino terminus of both peptides (Mermelstein, F. et al.(1996) Genes Dev. 10: 1033-1048). The heterodimer repressestranscription by binding to the TFIID-TATA promoter complex. Thisprecludes the association of TFIIA and/or TFIIB with the TFIID-TATApromoter complex and prematurely terminates the assembly of thetranscription initiation complex.

The discovery of a new transcription repressor similar to NC2α and DRAP1and the polynucleotides which encode it satisfies a need in the art byproviding new compositions which are useful in diagnosing, preventing,and treating inflammation and disorders associated with cellproliferation and apoptosis.

SUMMARY OF THE INVENTION

The invention features a new transcription repressor hereinafterdesignated TRAREP and characterized as having similarity to the αsubunit of a human negative cofactor 2, NC2α, and a human Dr1-associatedcorepressor, DRAP1.

Accordingly, the invention features a substantially purified TRAREPhaving the amino acid sequence shown in SEQ ID NO: 1.

One aspect of the invention features an isolated and substantiallypurified polynucleotide that encodes TRAREP. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO: 2.

The invention also relates to a polynucleotide sequence comprising thecomplement of SEQ ID NO: 2 or variants thereof. In addition, theinvention features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO: 2.

The invention additionally features nucleic acid sequences encodingfragments of the polynucleotides encoding TRAREP, or the complement ofthe polynucleotide sequences, as well as expression vectors and hostcells comprising polynucleotides that encode TRAREP. The invention alsofeatures antibodies which bind specifically to TRAREP, andpharmaceutical compositions comprising substantially purified TRAREP.The invention also features methods for stimulating cell proliferationand for treating or preventing a disorder associated with apoptosis byadministering an antagonist of TRAREP, and for treating or preventinginflammation or a disorder associated with cell proliferation orapoptosis by administering TRAREP.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO: 1) and nucleicacid sequence (SEQ ID NO: 2) of TRAREP. The alignment was produced usingMACDNASIS PRO software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

FIG. 2 shows the amino acid sequence alignments among TRAREP (SEQ IDNO: 1) and the α subunit of a human negative cofactor 2, NC2α (GI1491710; SEQ ID NO: 3) and a human Dr1-associated corepressor, DRAP1 (GI1244714; SEQ ID NO: 4). The alignment was produced using themultisequence alignment program of LASERGENE software (DNASTAR Inc,Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots (MACDNASIS PRO software)for TRAREP (SEQ ID NO: 1) and DRAP1 (SEQ ID NO: 3), respectively. Thepositive X axis reflects amino acid position, and the negative Y axis,hydrophobicity.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

"Nucleic acid sequence" as used herein refers, to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,"amino acid sequence" as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragments or portionsthereof, and to naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8: 53-63).

TRAREP, as used herein, refers to the amino acid sequences ofsubstantially purified TRAREP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEWFragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of TRAREP, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic TRAREP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to TRAREP, causes a change in TRAREP which modulates the activityof TRAREP. Agonists may include proteins, nucleic acids, carbohydrates,or any other molecules which bind to TRAREP.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to TRAREP, blocks or modulates the biologicalor immunological activity of TRAREP. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to TRAREP.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of TRAREP. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of TRAREP.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of TRAREP orportions thereof and, as such, is able to effect some or all of theactions related to the transcription repressor.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding TRAREP or the encoded TRAREP.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification" as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO: 1" encompassesthe full-length human TRAREP and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding TRAREPor fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO: 2 by northern analysis is indicative ofthe presence of mRNA encoding TRAREP in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

"Alterations" in the polynucleotide of SEQ ID NO: 2, as used herein,comprise any alteration in the sequence of polynucleotides encodingTRAREP including deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodesTRAREP (e.g., by alterations in the pattern of restriction fragmentlength polymorphisms capable of hybridizing to SEQ ID NO: 2), theinability of a selected fragment of SEQ ID NO: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding TRAREP (e.g., using fluorescent in situhybridization FISH! to metaphase chromosome spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind TRAREPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

The invention is based on the discovery of a new transcription repressor(TRAREP), the polynucleotides encoding TRAREP, and the use of thesecompositions for the diagnosis, prevention, or treatment of inflammationand disorders associated with cell proliferation and apoptosis.

Nucleic acids encoding the human TRAREP of the present invention werefirst identified in Incyte Clone 79591 from a rheumatoid synovium tissuecDNA library (SYNORAB01) through a computer search for amino acidsequence alignments. A consensus sequence, SEQ ID NO: 2, was derivedfrom the following overlapping and/or extended nucleic acid sequences:Incyte Clones 981794 (TONGTUT01), 414075 (BRSTNOT01), 79591 (SYNORAB01),1620089 (BRAITUT01), and 882708 (THYRNOT02).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1, as shown in FIGS. 1A and 1B.TRAREP is 195 amino acids in length. TRAREP has a histone foldencompassing residues 13 to 77, one internal acidic motif encompassingresidues 135-154, one proline-rich motif encompassing residues 158-194,and an amino-terminal acidic domain encompassing residues 195-205.TRAREP also has one potential cAMP- and cGMP-dependent protein kinasephosphorylation site at K131-S134, eight potential casein kinase IIphosphorylation sites at T23-E26, S89-D92, S 134-D137, T136-E139,S138-E141, T147-D140, T149-E152, and S193-D196, and one potentialprotein kinase C phosphorylation site at S3-K5. As shown in FIG. 2,TRAREP has chemical and structural homology with the α subunit ofnegative cofactor, NC2α (GI 1491710; SEQ ID NO: 3) and theDr1-associated corepressor, DRAP1 (GI 1244714; SEQ ID NO: 4). Inparticular, TRAREP shares 100% and 93% identity with NC2α and DRAP1,respectively. As illustrated by FIGS. 3A and 3B, TRAREP and DRAP1 haverather similar hydrophobicity plots. Northern analysis shows theexpression of TRAREP in various cDNA libraries, at least 53% of whichare immortalized or cancerous, at least 21% of which involve immuneresponse, and at least 13% of which involve fetal/infant tissues ororgans.

The invention also encompasses TRAREP variants which retain thebiological or other functional activity of TRAREP. A preferred TRAREPvariant is one having at least 80%, and more preferably 90%, amino acidsequence identity to the TRAREP amino acid sequence (SEQ ID NO: 1). Amost preferred TRAREP variant is one having at least 95% amino acidsequence identity to SEQ ID NO: 1.

The invention also encompasses polynucleotides which encode TRAREP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of TRAREP can be used to generate recombinant molecules whichexpress TRAREP. In a particular embodiment, the invention encompassesthe polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 2as shown in FIGS. 1A and 1B.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding TRAREP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring TRAREP, and all such variations are tobe considered as being specifically disclosed.

Although nucleotide sequences which encode TRAREP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring TRAREP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding TRAREP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding TRAREP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode TRAREP and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding TRAREP or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO: 2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152: 399-407)and Kimmel, A. R. (1987; Methods Enzymol. 152: 507-511), and may be usedat a defined stringency.

Altered nucleic acid sequences encoding TRAREP which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent TRAREP. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentTRAREP. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of TRAREP is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding TRAREP. As used herein, an "allele" or "allelicsequence" is an alternative form of the gene which may result from atleast one mutation in the nucleic acid sequence. Alleles may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding TRAREP may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2: 318-322). In particular, genomic DNA is first amplified inthe presence of primer to linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16: 8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1: 111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19: 3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries (Clontech, Palo Alto, Calif.) to walk genomic DNA. Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode TRAREP, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of TRAREP in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressTRAREP.

As will be understood by those of skill in the art, it may beadvantageous to produce TRAREP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter TRAREPencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding TRAREP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of TRAREP activity, it may be useful toencode a chimeric TRAREP protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the TRAREP encoding sequenceand the heterologous protein sequence, so that TRAREP may be cleaved andpurified away from the heterologous moiety.

In another embodiment, sequences encoding TRAREP may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of TRAREP, or a portion thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using theABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of TRAREP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active TRAREP, the nucleotidesequences encoding TRAREP or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding TRAREP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding TRAREP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding TRAREP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for TRAREP. For example, when largequantities of TRAREP are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding TRAREP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264: 5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153: 516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding TRAREP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6: 307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R.et al. (1984) Science 224: 838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17: 85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

An insect system may also be used to express TRAREP. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding TRAREP may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of TRAREP will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which TRAREP may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91: 3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding TRAREP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing TRAREP in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding TRAREP. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding TRAREP, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressTRAREP may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11: 223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22: 817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150: 1-14);and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55: 121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding TRAREP isinserted within a marker gene sequence, recombinant cells containingsequences encoding TRAREP can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding TRAREP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding TRAREP and express TRAREP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding TRAREP can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding TRAREP. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding TRAREP to detect transformantscontaining DNA or RNA encoding TRAREP. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofTRAREP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TRAREP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding TRAREP includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding TRAREP, orany portions thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio)).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding TRAREP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeTRAREP may be designed to contain signal sequences which directsecretion of TRAREP through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingTRAREP to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and TRAREP may be used to facilitate purification.One such expression vector provides for expression of a fusion proteincontaining TRAREP and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography) as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying TRAREP from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12: 441-453).

In addition to recombinant production, fragments of TRAREP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154). Proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments ofTRAREP may be chemically synthesized separately and combined usingchemical methods to produce the full length molecule.

THERAPEUTICS

TRAREP shares chemical and structural homology with a two humantranscription repressors, NC2α (GI 1491710) and DRAP1 (GI 1244714).Northern analysis shows that the expression of TRAREP is associated withcell proliferation, inflammation, and fetal development.

Decreased expression of TRAREP appears to be associated with increasedcell proliferation. Therefore, in one embodiment, TRAREP or a fragmentor derivative thereof may be administered to a subject to prevent ortreat a disorder associated with excessive cell proliferation. Suchdisorders include various types of cancer including, but not limited to,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and particularly, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus.

In another embodiment, an agonist of TRAREP or a derivative or fragmentthereof may be used to modulate the activity of TRAREP and to prevent ortreat a disorder associated with excessive cell proliferation including,but not limited to, those listed above.

In still another embodiment, a vector capable of expressing TRAREP, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with excessive cell proliferation including, but notlimited to, those listed above.

In another embodiment, TRAREP or a fragment or derivative thereof may beadministered to a subject to prevent or treat inflammation associatedwith any disorder including, but are not limited to, Addison's disease,adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma.

In another embodiment, an agonist of TRAREP or a fragment or derivativethereof may be used to modulate the activity of TRAREP and to prevent ortreat inflammation associated with any disorder including, but notlimited to, those listed above.

In still another embodiment, a vector capable of expressing TRAREP, or afragment or a derivative thereof, may be used to prevent or treatinflammation associated with any disorder including, but not limited to,those listed above.

Although increased expression of TRAREP and apoptosis are normal infetal development, the same expression levels appear to be associatedwith several disorders in which apoptosis is detrimental to the subject.Therefore, in one embodiment, an antagonist of TRAREP or a fragment orderivative thereof may be administered to a subject to prevent or treata disorder associated with apoptosis. Such disorders include, but arenot limited to, AIDS and other infectious or genetic immunodeficiencies,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, andcerebellar degeneration, myelodysplastic syndromes such as aplasticanemia, ischemic injuries such as myocardial infarction, stroke, andreperfusion injury, toxin-induced diseases such as alcohol-induced liverdamage, cirrhosis, and lathyrism, wasting diseases such as cachexia,viral infections such as by hepatitis B and C, and osteoporosis. In oneaspect, an antibody specific for TRAREP may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express TRAREP.

In another embodiment, a vector expressing the antisense orcomplementary sequence of the polynucleotide encoding TRAREP, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

In a further embodiment, an antagonist or an inhibitor of TRAREP, or afragment or a derivative thereof, may be added to cells to stimulatecell proliferation. In particular, an antagonist of TRAREP may be addedto a cell or cells in vivo using delivery mechanisms such as liposomes,viral based vectors, or electroinjection for the purpose of promotingregeneration or differentiation of the cell or cells. In addition, anantagonist of TRAREP may be added to a cell, cell line, tissue or organculture in vitro or ex vivo to stimulate cell proliferation for use inheterologous or autologous transplantation. In some cases, the cell willhave been selected for its ability to fight an infection or a cancer orto correct a genetic defect such as sickle cell anemia, β thalassemia,cystic fibrosis, or Huntington's chorea. In one aspect, an antibodyspecific for TRAREP may be used directly as an antagonist, or indirectlyas a targeting or delivery mechanism for bringing a pharmaceutical agentto cells or tissue which express TRAREP.

In a still further embodiment, a vector expressing the antisense orcomplementary sequence of the polynucleotide encoding TRAREP, or afragment or a derivative thereof, may be used to stimulate cellproliferation, as detailed above.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, complementary or antisense sequences or vectors ofthe invention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

Antagonists or inhibitors of TRAREP may be produced using methods whichare generally known in the art. In particular, purified TRAREP may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind TRAREP.

Antibodies to TRAREP may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith TRAREP or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to TRAREP have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of TRAREP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to TRAREP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256: 495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81: 31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81: 6851-6855; Neuberger, M. S. et al. (1984) Nature312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceTRAREP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88: 11120-3).

Antibodies specific for TRAREP may also be produced by inducing in vivoproduction in the lymphocyte population or by screening recombinantimmunoglobulin libraries or panels of highly specific binding reagentsas disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl.Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349: 293-299).

Antibody fragments which contain specific binding sites for TRAREP mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254: 1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between TRAREP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering TRAREP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingTRAREP, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding TRAREP may be used in situations in which it would be desirableto block the transcription of the mRNA. In particular, cells may betransformed with sequences complementary to polynucleotides encodingTRAREP. Thus, antisense molecules may be used to modulate TRAREPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding TRAREP.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingTRAREP. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding TRAREP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes TRAREP. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding TRAREP, i.e., the promoters, enhancers, andintrons. Oligonucleotides derived from the transcription initiationsite, e.g., between positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using "triple helix"base-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding TRAREP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding TRAREP. Such DNA sequences may be incorporatedinto a wide variety of vectors with suitable RNA polymerase promoterssuch as T7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of TRAREP, antibodies toTRAREP, mimetics, agonists, antagonists, or inhibitors of TRAREP. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0. 1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of TRAREP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example TRAREP or fragments thereof, antibodies ofTRAREP, agonists, antagonists or inhibitors of TRAREP, which amelioratesthe symptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind TRAREP may beused for the diagnosis of conditions or diseases characterized byexpression of TRAREP, or in assays to monitor patients being treatedwith TRAREP, agonists, antagonists or inhibitors. The antibodies usefulfor diagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for TRAREP includemethods which utilize the antibody and a label to detect TRAREP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringTRAREP are known in the art and provide a basis for diagnosing alteredor abnormal levels of TRAREP expression. Normal or standard values forTRAREP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to TRAREP under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of TRAREPexpressed in subject, control and disease samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingTRAREP may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofTRAREP may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofTRAREP, and to monitor regulation of TRAREP levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TRAREP or closely related molecules, may be used to identifynucleic acid sequences which encode TRAREP. The specificity of theprobe, whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding TRAREP, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe TRAREP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO: 2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring TRAREP.

Means for producing specific hybridization probes for DNAs encodingTRAREP include the cloning of nucleic acid sequences encoding TRAREP orTRAREP derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding TRAREP may be used for the diagnosisof disorders associated with the expression of TRAREP. Examples of suchdisorders include: various types of cancer such as adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, andparticularly, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; disorders associated with inflammation such as Addison'sdisease, adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma; and disordersassociated apoptosis such as AIDS and other infectious or geneticimmunodeficiencies, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, and cerebellar degeneration, myelodysplastic syndromes suchas aplastic anemia, ischemic injuries such as myocardial infarction,stroke, and reperfusion injury, toxin-induced diseases such asalcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseasessuch as cachexia, viral infections such as by hepatitis B and C, andosteoporosis. The polynucleotide sequences encoding TRAREP may be usedin Southern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered TRAREP expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequences encoding TRAREP may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingTRAREP may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding TRAREP in the sample indicates thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of TRAREP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes TRAREP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding TRAREP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5->3') and another with antisense(3'<-5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of TRAREPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159: 235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequences whichencode TRAREP may also be used to generate hybridization probes whichare useful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7: 127-134, and Trask, B. J.(1991) Trends Genet. 7: 149-154.

FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual ofBasic Techniques, Pergamon Press, New York, N.Y.) may be correlated withother physical chromosome mapping techniques and genetic map data.Examples of genetic map data can be found in the 1994 Genome Issue ofScience (265: 1981 f). Correlation between the location of the geneencoding TRAREP on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336: 577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, TRAREP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenTRAREP and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to TRAREP large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with TRAREP, or fragments thereof, and washed. Bound TRAREP isthen detected by methods well known in the art. Purified TRAREP can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding TRAREP specificallycompete with a test compound for binding TRAREP. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with TRAREP.

In additional embodiments, the nucleotide sequences which encode TRAREPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I SYNORAB01 cDNA Library Construction

The cDNA library SYNORAB01 was constructed using RNA isolated from thesynovial membrane tissue of a 68-year-old Caucasian female withrheumatoid arthritis. The frozen tissue was immediately homogenized andlysed using a Brinkmann Homogenizer Polytron-PT 3000 (BrinkmannInstruments, Inc., Westbury, N.Y.) in guanidinium isothiocyanatesolution. Lysates were then loaded on a 5.7M CsCl cushion andultracentrifuged in an SW28 swinging bucket rotor for 18 hours at 25,000rpm at ambient temperature. The RNA was extracted once with acid phenolat pH 4.0 and once with phenol chloroform at pH 8.0 and precipitatedusing 0.3M sodium acetate and 2.5 volumes of ethanol, resuspended inDEPC-treated water and DNase treated for 25 min at 37° C. The reactionwas stopped with an equal volume of acid phenol, and the RNA wasisolated with the OLIGOTEX kit (QIAGEN Inc, Chatsworth, Calif.) and usedto construct the cDNA library.

Poly-A⁺ mRNA was isolated using a biotinylated oligo d(T) primer andstreptavidin coupled to a paramagnetic particle (Promega, Madison,Wis.). The cDNA library was custom prepared by Stratagene using acombination of oligo d(T) and random priming. Synthetic adaptors wereligated to the blunt-ended cDNA population which was then ligated to thephage lambda-derived UNI-ZAP vector (Stratagene).

The cDNA libraries can be screened with either DNA probes or antibodyprobes and the PBLUESCRIPT phagemid (Stratagene) can be rapidly excisedin vivo. The custom-constructed library phage particles were transfectedinto E. coli host strain XL1-BLUE (Stratagene).

II Isolation and Sequencing of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which the host bacterial strain was coinfectedwith both the lambda library phage and an f1 helper phage. Polypeptides,or enzymes derived from both the library-containing phage and the helperphage nicked the lambda DNA, initiated new DNA synthesis from definedsequences on the lambda target DNA and created a smaller, singlestranded circular phagemid DNA molecule that included all DNA sequencesof the PBLUESCRIPT plasmid and the cDNA insert. The phagemid DNA wassecreted from the cells, purified, and used to re-infect fresh hostcells where the double stranded phagemid DNA was produced. Because thephagemid carried the gene for β-lactamase, the newly transformedbacteria were selected on medium containing ampicillin.

Plasmid DNA was purified using the MAGIC MINIPREPS DNA purificationsystem (Catalogue #A7100; Promega). The DNA was eluted from thepurification resin already prepared for DNA sequencing and otheranalytical manipulations. Phagemid DNA was also purified using theQIAWELL-8, QIAWELL PLUS and QIAWELL ULTRA DNA Purification System(QIAGEN, Chatsworth, Calif.). The DNA was eluted from the purificationresin and prepared for DNA sequencing and other analyticalmanipulations.

The cDNAs were sequenced by the method of Sanger F and A R Coulson(1975; J Mol Biol 94: 441f), using a Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.) in combination with four Peltier Thermal Cyclers (PTC200; MJResearch, Watertown, Mass.) and Applied Biosystems 373 DNA SequencingSystems (Perkin Elmer), and reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

After the reading frame was determined, the nucleotide sequences of theSequence Listing or amino acid sequences deduced from them were used asquery sequences against databases such as GenBank, SwissProt, BLOCKS,and Pima II. These databases which contain previously identified andannotated sequences, were searched for regions of homology (similarity)using BLAST, which stands for Basic Local Alignment Search Tool(Altschul (1993) supra, Altschul (1990) supra).

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith et al. (1992, Protein Engineering 5: 35-51),incorporated herein by reference, could have been used when dealing withprimary sequence patterns and secondary structure gap penalties. Thesequences disclosed in this application have lengths of at least 49nucleotides, and no more than 12% uncalled bases (where N is recordedrather than A, C, G, or T).

The BLAST approach, as detailed in Karlin et al. (supra) andincorporated herein by reference, searched for matches between a querysequence and a database sequence. BLAST evaluated the statisticalsignificance of any matches found, and reported only those matches thatsatisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding TRAREP occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of TRAREP-Encoding Polynucleotides

Nucleic acid sequence of Incyte clone 79591 or SEQ ID NO: 2 is used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length or for obtaining 5' or 3', intron or othercontrol sequences from genomic libraries. One primer is synthesized toinitiate extension in the antisense direction (XLR) and the other issynthesized to extend sequence in the sense direction (XLF). Primers areused to facilitate the extension of the known sequence "outward"generating amplicons containing new, unknown nucleotide sequence for theregion of interest. The initial primers are designed from the cDNA usingOLIGO 4.06 (National Biosciences), or another appropriate program, to be22-30 nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M. J. Research,Watertown, Mass.) and the following parameters:

Step 1 94° C. for 1 min (initial denaturation)

Step 2 65° C. for 1 min

Step 3 68° C. for 6 min

Step 4 94° C. for 15 sec

Step 5 65° C. for 1 min

Step 6 68° C. for 7 min

Step 7 Repeat step 4-6 for 15 additional cycles

Step 8 94° C. for 15 sec

Step 9 65° C. for 1 min

Step 10 68° C. for 7:15 min

Step 11 Repeat step 8-10 for 12 cycles

Step 12 72° C. for 8 min

Step 13 4° C. (and holding)

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as, QIAQUICK (QIAGEN Inc., Chatsworth, Calif.). After recovery ofthe DNA, Klenow enzyme is used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

Step 1 94° C. for 60 sec

Step 2 94° C. for 20 sec

Step 3 55° C. for 30 sec

Step 4 72° C. for 90 sec

Step 5 Repeat steps 2-4 for an additional 29 cycles

Step 6 72° C. for 180 sec

Step 7 4° C. (and holding)

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO: 2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of γ-³² P! adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with SEPHADEX G-25superfine resin column (Pharmacia & Upjohn). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense or Complementary Sequences

Antisense molecules or nucleic acid sequences complementary to theTRAREP-encoding sequence, or any part thereof, are used to inhibit invivo or in vitro expression of naturally occurring TRAREP. Although useof antisense oligonucleotides, comprising about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. An oligonucleotide based on the coding sequencesof TRAREP, as shown in FIGS. 1A and 1B, is used to inhibit expression ofnaturally occurring TRAREP. The complementary oligonucleotide isdesigned from the most unique 5' sequence as shown in FIGS. 1A and 1Band used either to inhibit transcription by preventing promoter bindingto the upstream nontranslated sequence or translation of anTRAREP-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the signal and 5' sequence of SEQ ID NO:2, an effective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the signal or 5' codingsequence of the polypeptide as shown in FIGS. 1A and 1B.

VIII Expression of TRAREP

Expression of TRAREP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, PSPORT1, previously used for thegeneration of the cDNA library is used to express TRAREP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofTRAREP into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of TRAREP Activity

TRAREP can be expressed by transforming a mammalian cell line such asCOS7, HeLa or CHO with an eukaryotic expression vector encoding TRAREP.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. The cells are incubated for 48-72 hours after transformationunder conditions appropriate for the cell line to allow expression ofTRAREP. Then, phase microscopy is used to compare the mitotic index oftransformed versus control cells. A decrease in the mitotic indexindicates TRAREP activity.

X Production of TRAREP Specific Antibodies

TRAREP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO: 2 is analyzed using LASERGENEsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring TRAREP Using Specific Antibodies

Naturally occurring or recombinant TRAREP is substantially purified byimmunoaffinity chromatography using antibodies specific for TRAREP. Animmunoaffinity column is constructed by covalently coupling TRAREPantibody to an activated chromatographic resin, such as CnBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing TRAREP is passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of TRAREP (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/TRAREP binding (e.g., a buffer of pH 2-3 or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andTRAREP is collected.

XII Identification of Molecules Which Interact with TRAREP

TRAREP or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled TRAREP, washed and any wells withlabeled TRAREP complex are assayed. Data obtained using differentconcentrations of TRAREP are used to calculate values for the number,affinity, and association of TRAREP with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 205 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: SYNORAB01    (B) CLONE: 1620089    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetProSerLysLysLysLysTyrAsnAlaArgPheProProAlaArg    151015    IleLysLysIleMetGlnThrAspGluGluIleGlyLysValAlaAla    202530    AlaValProValIleIleSerArgAlaLeuGluLeuPheLeuGluSer    354045    LeuLeuLysLysAlaCysGlnValThrGlnSerArgAsnAlaLysThr    505560    MetThrThrSerHisLeuLysGlnCysIleGluLeuGluGlnGlnPhe    65707580    AspPheLeuLysAspLeuValAlaSerValProAspMetGlnGlyAsp    859095    GlyGluAspAsnHisMetAspGlyAspLysGlyAlaArgArgGlyArg    100105110    LysProGlySerGlyGlyArgLysAsnGlyGlyMetGlyThrLysSer    115120125    LysAspLysLysLeuSerGlyThrAspSerGluGlnGluAspGluSer    130135140    GluAspThrAspThrAspGlyGluGluGluThrSerGlnProProPro    145150155160    GlnAlaSerHisProSerAlaHisPheGlnSerProProThrProPhe    165170175    LeuProPheAlaSerThrLeuProLeuProProAlaProProGlyPro    180185190    SerAlaProAspGluGluAspGluGluAspTyrAspSer    195200205    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 737 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: SYNORAB01    (B) CLONE: 1620089    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CGGCGCTGGACCCNACGCGGCGAGAGAGGCCCCGAGATGCCGAGCAAGAAGAAGAAGTAC60    AACGCGCGGTTCCCGCCGGCGCGGATCAAGAAGATCATGCAGACGGACGAAGAGATTGGG120    AAGGTGGCGGCGGCGGTGCCTGTCATCATCTCCCGGGCGCTCGAGCTCTTCCTAGAGTCG180    CTGTTGAAGAAGGCCTGCCAGGTGACCCAGTCGCGGAACGCGAAGACCATGACCACATCC240    CACCTGAAGCAGTGCATCGAGCTGGAGCAGCAGTTTGACTTCTTGAAGGACCTGGTGGCA300    TCKGTTCCCGACATGCAGGGGGACGGGGAAGACAACCACATGGATGGGGACAAGGGCGCC360    CGCAGRGGCCGGAAGCCAGGCAGCGGCGGCCGGAAGAACGGTGGGATGGGAACGAAAAGC420    AAGGACAAGAAGCTKTCCGGGACAGACTCGGAGCAGGAGGATGAATCTGAGGACACAGAT480    ACTGATGGGGAAGAGGAGACATCACARCCCCCACCCCAGGCCAGCCACCCCTCTGCCCAC540    TTTCAGAGCCCCCCGACAACCTTCCTGCCCTTCGCCTCTACTCTGCCTTTGCCCCCAGCG600    CCCCCGGGCCCCTCAGCACCTGATGAAGAGGACGAAGAAGATTACGACTCCTAGCGCCTT660    CTGCCCCCCAGACCATAGCCCCTTTTAGTTGGTTTTAGTTGCTCTGGGGGGAGGAGAGAA720    GGTAGAGCTGTTCTTAA737    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 205 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: GenBank    (B) CLONE: 1491710    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    MetProSerLysLysLysLysTyrAsnAlaArgPheProProAlaArg    151015    IleLysLysIleMetGlnThrAspGluGluIleGlyLysValAlaAla    202530    AlaValProValIleIleSerArgAlaLeuGluLeuPheLeuGluSer    354045    LeuLeuLysLysAlaCysGlnValThrGlnSerArgAsnAlaLysThr    505560    MetThrThrSerHisLeuLysGlnCysIleGluLeuGluGlnGlnPhe    65707580    AspPheLeuLysAspLeuValAlaSerValProAspMetGlnGlyAsp    859095    GlyGluAspAsnHisMetAspGlyAspLysGlyAlaArgArgGlyArg    100105110    LysProGlySerGlyGlyArgLysAsnGlyGlyMetGlyThrLysSer    115120125    LysAspLysLysLeuSerGlyThrAspSerGluGlnGluAspGluSer    130135140    GluAspThrAspThrAspGlyGluGluGluThrSerGlnProProPro    145150155160    GlnAlaSerHisProSerAlaHisPheGlnSerProProThrProPhe    165170175    LeuProPheAlaSerThrLeuProLeuProProAlaProProGlyPro    180185190    SerAlaProAspGluGluAspGluGluAspTyrAspSer    195200205    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 193 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: GenBank    (B) CLONE: 1244714    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetGluPheArgIleLysLysIleMetGlnThrAspGluGluIleGly    151015    LysValAlaAlaAlaValProValIleIleSerArgAlaLeuGluLeu    202530    PheLeuGluSerLeuLeuLysLysAlaCysGlnValThrGlnSerArg    354045    AsnAlaLysThrMetThrThrSerHisLeuLysGlnCysIleGluLeu    505560    GluGlnGlnPheAspPheLeuLysAspLeuValAlaSerValProAsp    65707580    MetGlnGlyAspGlyGluAspAsnHisMetAspGlyAspLysGlyAla    859095    ArgArgGlyArgLysProGlySerGlyGlyArgLysAsnGlyGlyMet    100105110    GlyThrLysSerLysAspLysLysLeuSerGlyThrAspSerGluGln    115120125    GluAspGluSerGluAspThrAspThrAspGlyGluGluGluThrSer    130135140    GlnProProProGlnAlaSerHisProSerAlaHisPheGlnSerPro    145150155160    ProThrProPheLeuProPheAlaSerThrLeuProLeuProProAla    165170175    ProProGlyProSerAlaProAspGluGluAspGluGluAspTyrAsp    180185190    Ser    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequenceencoding the human transcription repressor of SEQ ID NO:
 1. 2. Apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of claim
 1. 3. A composition comprising thepolynucleotide sequence of claim 1 and a pharmaceutically acceptablecarrier.
 4. An isolated and purified polynucleotide sequence consistingof SEQ ID NO:
 2. 5. A polynucleotide sequence which is complementary tothe polynucleotide sequence of claim
 4. 6. A composition comprising thepolynucleotide sequence of claim 5 and a pharmaceutically acceptablecarrier.
 7. An expression vector containing the polynucleotide sequenceof claim
 1. 8. A host cell containing the vector of claim
 7. 9. A methodfor producing a polypeptide comprising the amino acid sequence of SEQ IDNO: 1, or fragments thereof, the method comprising the steps of:a)culturing the host cell of claim 8 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.